Site-specific mutagenesis inLegionella pneumophilaby allelic exchange using counterselectable ColE1 vectors

To study the molecular pathogenesis of infection by Legionella pneumophila, a technique of site-specific mutagenesis by allelic exchange was evaluated. To develop this system, we optimized conjugal DNA transfer by isolating a mutant that functions 1000-fold more efficiently as a recipient than the wild type strain, identified two counter-selectable markers, rpsL and sacB, that function in L. pneumophila, and constructed a counterselectable Co1E1 vector. Allelic exchange of a L. pneumophila chrosomal gene was achieved at a frequency of 10−5 per transconjugant. The allelic exchange procedure itself did not alter the ability of L. pneumophila to infect macrophages, indicating that the system can be used to study this aspect of virulence.